This application is based upon and claims the benefit of priority from Japanese Patent Application No. 2003-26303, filed on Feb. 3, 2003; the entire contents of this application are incorporated herein by reference.
The present invention is directed to a method for detecting a DNA strand break using a PprA protein derived from Deinococcus radiodurans which recognizes and binds to a DNA strand break. The present invention is also directed to a kit for detecting one or more DNA strand break which comprises an amount of PprA protein derived from Deinococcus radiodurans which recognize and bind to one or more DNA strand break.
It is essential for continued existence of species that DNA, a carrier of genetic information, be stably maintained, and transferred to progeny. However, living organisms are continually exposed to environmental factors which trigger damage to DNA. Such factors consist of extrinsic such as radiation, ultraviolet rays, agents, or other mutagens which are present in foods and in the environment in general, and of intrinsic factors such as active oxygen, which is generated in the process of metabolism, and also error(s) in DNA replication. Such DNA damage inhibits the replication or transcription of DNA, resulting in mutation which may cause cell death, aging, tumorigenesis or carcinogenesis.
A DNA strand break is a particularly serious form of DNA damage and has an especially deleterious effect on cells. Therefore, from the viewpoint of risk assessment, it is believed to be important to examine a frequency of generation of DNA strand break(s), a distribution of DNA strand break(s) in cells, and differences in susceptibility between DNA strand break(s) and other DNA damage. However, to date there has existed no known sufficiently sensitive method for directly detecting a life-threatening DNA strand break. This was resulted in a delay in technical progress in the art. Therefore, it is important for technical progress in the field relating to DNA damage and DNA repair of higher organisms to be able to directly examine a distribution of and repair process of DNA strand break(s), through the development of an in situ method of visualizing DNA damage and DNA repair.
To date, several examples are known for detecting DNA strand break(s) using DNA binding protein and antibodies which specifically bind thereto in mammalian cells such as follows: one example it to use, in B lymphocytes, Nbs1 protein, Rad51 protein, Brcal protein, or gamma-H2AX protein, and antibodies which specifically bind to these proteins to detect DNA strand break(s) (Petersen et al., Nature, 414: 660-665, 2001); and another example is to use, in human fibroblast cells, Mre11 protein or gamma-H2AX protein and antibodies which specifically bind to these proteins to detect DNA strand break(s) (Limoli et al., Proc. Natl. Acad. Sci. USA, 99: 233-238, 2002).
However, since endogenous DNA binding proteins are used in these methods for detecting DNA strand break(s), these methods have disadvantages that DNA strand break(s) cannot be detected in a sample which is prepared from an organism not having such DNA binding proteins, and that it is difficult to detect initial damage of DNA because it takes a certain period to bind DNA binding protein to DNA strand break(s).
The present invention is directed to a method for directly measuring the in vivo distribution and the frequency of generation of DNA strand break(s) which triggers cell death and mutation. More specifically, the present invention is directed to a method for detecting a DNA strand break using a Ppra protein derived from Deinococcus radiodurans which has an ability to recognize and bind to a DNA strand break, well as a kit for detecting a DNA strand break comprising an amount of Ppra protein.